Nitric oxide inhibits FTO demethylase activity to regulate N⁶-methyladenosine mRNA methylation

N⁶-methyladenosine (m⁶A) is the most abundant internal modification on eukaryotic mRNAs. Demethylation of m⁶A on mRNA is catalyzed by the enzyme fat mass and obesity-associated protein (FTO), a member of the nonheme Fe(II) and 2-oxoglutarate (2-OG)-dependent family of dioxygenases. FTO activity and m⁶A-mRNA are dysregulated in multiple diseases including cancers, yet endogenous signaling molecules that modulate FTO activity have not been identified. Here we show that nitric oxide (NO) is a potent inhibitor of FTO demethylase activity by directly binding to the catalytic iron center, which causes global m⁶A hypermethylation of mRNA in cells and results in gene-specific enrichment of m⁶A on mRNA of NO-regulated transcripts. Both cell culture and tumor xenograft models demonstrated that endogenous NO synthesis can regulate m⁶A-mRNA levels and transcriptional changes of m⁶A-associated genes. These results build a direct link between NO and m⁶A-mRNA regulation and reveal a novel signaling mechanism of NO as an endogenous regulator of the epitranscriptome.